Ultra-high throughput-based screening for the discovery of antiplatelet drugs affecting receptor dependent calcium signaling dynamics

Distinct platelet activation patterns are elicited by the tyrosine kinase-linked collagen receptor glycoprotein VI (GPVI) and the G-protein coupled protease-activated receptors (PAR1/4) for thrombin. This is reflected in the different platelet Ca2+ responses induced by the GPVI agonist collagen-related peptide (CRP) and the PAR1/4 agonist thrombin. Using a 96 well-plate assay with human Calcium-6-loaded platelets and a panel of 22 pharmacological inhibitors, we assessed the cytosolic Ca2+ signaling domains of these receptors and developed an automated Ca2+ curve algorithm. The algorithm was used to evaluate an ultra-high throughput (UHT) based screening of 16,635 chemically diverse small molecules with orally active physicochemical properties for effects on platelets stimulated with CRP or thrombin. Stringent agonist-specific selection criteria resulted in the identification of 151 drug-like molecules, of which three hit compounds were further characterized. The dibenzyl formamide derivative ANO61 selectively modulated thrombin-induced Ca2+ responses, whereas the aromatic sulfonyl imidazole AF299 and the phenothiazine ethopropazine affected CRP-induced responses. Platelet functional assays confirmed selectivity of these hits. Ethopropazine retained its inhibitory potential in the presence of plasma, and suppressed collagen-dependent thrombus buildup at arterial shear rate. In conclusion, targeting of platelet Ca2+ signaling dynamics in a screening campaign has the potential of identifying novel platelet-inhibiting molecules.


Flow cytometric platelet viability assay
Washed platelets (50 × 10 9 /L) in Hepes buffer pH 7.45 were pre-incubated for 10 min at room temperature with selected compounds or vehicle control (<0.5% DMSO, f.c.).Subsequently, the platelets were loaded with calcein-AM (20 µM) for 20 min at 37 ºC.The de-esterified calcein that is accumulated and retained into the cytosol is a common viability marker 2 .Triplicate samples of the calcein-loaded platelets were measured for mean fluorescence intensity, using an Accuri C6 flow cytometer (BD Bioscience, Franklin Lakes, NJ, USA).Triplicate samples per condition were analyzed.The threshold level for non-toxicity wase set at 10% DMSO.

Figure S4
. Platelet Calcium-6 fluorescence traces with selected small molecules during initial screening.Calcium-6 loaded platelets (4 µL, 400 × 10 9 /L) in 1536-well plates were stimulated with CRP 10 µg/mL (a) or thrombin 4 nM (b) after preincubation with indicated compound (10 µM).Fluorescence changes from wells were recorded using a FLIPR-Tetra machine.Baseline fluorescence immediately before agonist addition was defined as F0.Of note: Optimal mixing was achieved by the injection speed, but the injection of a relatively large volume of agonist solution as well as light pathway interference of the tip resulted in a drop in fluorescence.

Figure S1 .
Figure S1.Ultrahigh throughput cytosolic Ca 2+ trace profiling.(a) Representative fluorescence rises of Calcium-6 loaded platelets induced by CRP or thrombin in 1,536-well plates, recorded by FLIPR-Tetra.Color index: green dot = compound addition; purple dot = agonist addition; green line = median level before compound addition; purple line = median basal level before agonist addition (parameter P1); red dot = peak level (P4); red line = interpolation of slope 1 (P3); blue line = interpolation of slope 2 (P5); grey line = interpolation of slope 3 (P6); cyan line = fluorescence between addition of compound and agonist as a reference.Parameters area under the curve AUC (P7), and maximal cytosolic Ca 2+ increase (P2) relative to the basal level.(b-c), Calcium-6 loaded platelets in 96-or 1,536-well plates were stimulated with CRP (10 µg/mL) or thrombin (4 nM).The platelets in wells were preincubated with one of 22 inhibitors (prescreening) or 16,635 small molecules (screening), as indicated.Shown are Spearman correlation matrices for the various curve parameters per agonist (CRP or thrombin) and per (pre)screening dataset.(b) Comparison of effects of 22 reference compounds in 96-well plate.(c), Comparison of effects of 16,635 small molecules in 1536-well plates.Red and blue colors indicate negative and positive correlations, respectively.

Figure S2 .
Figure S2.Physicochemical characteristics of compounds in the SMC library and of the selected effective compounds.Shown are physicochemical characteristics in the employed SMC library of 15,355 small molecule compounds (a) of 102 selected compounds for CRP-stimulated platelets (b) and of 38 compounds for thrombin-stimulated platelets (c) (2 nd selection round).Characteristics are indicated of molecular weight; lipophilic partition coefficient (LogP); numbers of hydrogen bond acceptors (HBA) and donors (HBD); topological polar surface area of nitrogen, oxygen, phosphate and sulfur atoms (TPSA); heavy atom count (HAC) for total number of non-hydrogen atoms; and degree of carbon saturation (Fsp3).

Figure S3 .
Figure S3.Distribution profiles of summed Z-scores per compound library and per agonist.The Prestwick Chemical library and the larger SMC library of small molecules were screened on effects of platelets stimulated with CRP (a) or thrombin (b).Seven parameters (P1-7) were derived from the cytosolic Ca 2+ traces.Shown are stacked histograms of Z-scores for each parameter and compound.Dashed lines indicate Z-score threshold of > | 4 |.

Figure S8 .
Figure S8.Prediction analysis of signaling pathways inhibited by ethopropazine.(a), K-means, principal component analysis (PCA) and cluster analysis combining effects of 22 reference compounds and ethopropazine (10 and 30 µM, in bold) on CRP-induced Ca 2+ rises in platelets (curve parameters P1-6).(b), Euclidean distance matrix of k-means, indicating per reference compound high (red) or low (blue) similarity of altered [Ca 2+ ]i curves in comparison to ethopropazine effects.

Table S1 .
Characteristics of the reference inhibitor panel.Indicated are known targets of the 22 reference platelet inhibitors, links to UniProtKD, and reported effects on CRP-or thrombin-induced [Ca 2+ ]i traces in platelets.For further characterization of the effects and statistical analysis, see Suppl.Datafile 1.

Table S2 . Reactome pathway analysis of targets of effective inhibitors of CRP-and thrombin-induced Ca 2+ rises in platelets.
Indicated are per agonist, the Reactome pathway identifiers; pathway names; numbers and ratios of entities identified; P-values; false discovery rates (FDR), thrombin (Thr).